Extensive posttranscriptional processing is required before eukaryotic pre-mRNA matures and exits from the nucleus to the cytoplasm, including the addition of a 7-methylguanosine cap at the 5′ end, the cleavage and addition of a poly-A tail at the 3′ end as well as the removal of intervening sequences or introns by the spliceosome. The vast majority of higher eukaryotic genes contain multiple introns that need to be spliced out with high precision and fidelity in order to maintain the reading frame of the exons. Splicing of pre-mRNA depends on the recognition of short consensus sequences at the boundaries and within introns by an array of small nuclear ribonucleoprotein (snRNP) complexes (consisting of snRNPs U1, U2, U4, U5, U6, U11, U12m U4atc and U6atc) and a large number of proteins, including spliceosomal proteins and positively as well as negatively acting splicing modulators (Black, 2003, Annu Rev Biochem 72:291-336; Faustino et al., 2003, Genes Dev 17:419-437; Graveley, 2006, RNA 6:1197-1211). Serine-arginine-rich (SR)-domain-containing proteins (Manley et al., 1996, Genes Dev 10:1569-1579) generally serve to promote constitutive splicing. They also modulate alternative splicing by binding to intronic or exonic splicing enhancer (ISE or ESE, respectively) sequences (Graveley, 2000, RNA 6:1197-1211; Black, 2003, Annu Rev Biochem 72:291-336). Other pre-mRNA binding proteins that lack SR domains, such as hnRNPs, regulate splicing by binding to intronic or exonic splicing suppressor (ISS or ESS, respectively) sites and also act as general splicing modulators (Dreyfuss et al., 2002, Nat Rev Mol Cell Biol 3:195-205; Wang and Burge et al., 2008, RNA 14:802-813).
The SR protein family is a class of at least 10 proteins that have a characteristic serine/arginine rich domain in addition to an RNA-binding region (Bourgeois et al., 2004, frog Nucleic Acid Res Mol Biol 78:37-88). SR proteins are generally thought to enhance splicing by simultaneously binding to U170K, a core component of the U1 snRNP, at the 5′ splice site, and the U2AF35 at the 3′ splice site, thus bridging the two ends of the intron (Jamison et al., 1995, Nucleic Acids Res 23:3260-3267; Katz et al., 1994, Nature 368:119-124). While this particular function of SR proteins seems to be redundant, as any individual SR protein can commit a pre-mRNA for constitutive splicing, the role of the various SR proteins in alternative splicing of specific pre-mRNAs is distinct due in part to their ability to recognize and bind to unique consensus sequences (Bourgeois et al., 2004, Prog Nucleic Acid Res Mol Biol 78:37-88). Phosphorylation of the RS domain of SR proteins can lead to the regulation of their protein interactions, RNA binding, localization, trafficking, and role in alternative splicing (Caceres, et al., 1998, Genes Dev 12:55-66; Cao et al., 1997, RNA 3:1456-1467; Duncan et al., 1997, Mol Cell Biol 17:5996-6001; Misteli et al., 1998, J Cell Biol 143:297-307; Xiao et al., 1997, Genes Dev 11:334-344). Several cellular kinases that phosphorylate SR proteins have been identified, including SR protein Kinase (SRPKs) (Gui et al., 1994, Nature 369:678-682; Kuroyanagi et al., 1998, Biochem Biophys Res Commun 242:357-364), Cdc2-like kinases (Clks) (Ben-David et al., 1991, EMBO J. 10:317-325; Colwill et al., 1996, EMBO J 15:265-275), pre-mRNA processing mutant 4 (PRP4) (Kojima et al., 2001, J Biol Chem 276:32247-32256), and topoisomerase I (Rossi et al., 1996, Nature 381:80-82), Optimal phosphorylation of SR proteins is required for proper functioning as both hypo- and hyperphosphorylation of the RS domains is detrimental to their role in constitutive and alternative splicing (Prasad et al., 1999, Mol Cell Biol 19:6991-7000).
Besides its essential role in removing introns, splicing imprints the mRNA with a dynamic complex that is deposited around 20 nucleotides upstream of the exon-exon junction (Dostie et al., 2002, Curr Biol 12:1060-1067; Kataoka et al., 2004, J Biol Chem 279:7009-7013; Lau et al., 2003, Curr Biol 13:933-941; Tange et al., 2004, Curt Opin Cell Biol 16: 279-284). The exon junction complex (EJC) plays diverse roles in downstream mRNA biogenesis such as export to the cytoplasm, localization, non-sense mediated decay (NMD) and translation (Diem et al., 2007, Nat Struct Mol Biol 14:1173-1179; Hachet et al., 2004, Nature 428:959-963; Le Hir et al., 2001, EMBO Rep 2:1119-1124; Le Hir et al., 2001, EMBO J. 20:4987-4997; Nott et al., 2004, Genes Dev 18:210-222; Wiegand et al., 2003, Proc Natl Acad Sci USA 100:11327-11332; Zhang d al., 2007, Proc Natl Acad Sci USA 104:11574-11579). Phosphorylation and possibly methylation of at least one component of the EJC, Y14, has been shown to regulate its interaction with other proteins involved in spliced mRNA biogenesis, but the signal that modulates these modifications has not been clearly identified (Hsu et al, 2005, J Biol Chem 280:34507-34512). On the other hand, the EJC-dependent phosphorylation of Upf1 has been shown to be critical for triggering NMD (Kashima et al., 2006, Genes Dev 20:355-367). Since upstream signaling seems to be essential for regulating components of the EJC and their function in spliced mRNA biogenesis, it is important to fully understand these signals and determine whether they modulate the expression of a subset of spliced mRNAs.
Aberrations in splicing due to mutations in the consensus sequences involved in exon-intron boundary recognition are responsible for up to 15% of inherited diseases (Krawezak et al., 1992, Hum Genet 90:41-54). In addition, defects in the splicing machinery itself due to the loss or gain of function of splicing factors and modulators are causes of a wide range of human ailments from cancer to neurodegenerative diseases (Garcia-Blanco et al., 2004, Nat Biotechnol 22:535-546; Licatalosi et al., 2006, Neuron 52:93-101; Venables, 2004, Cancer Res 64:7647-7654). Over the past few years, it has been established that both constitutive and alternative splicing are subject to regulation by upstream signaling pathways. This regulation is essential during development, in tissue specific expression of certain isoforms, during the cell cycle and in response to extrinsic signaling molecules (Hagiwara, 2005, Biochim Biophys Acta 1754:324-331; Schwerk et al., 2005, Mol Cell 19:1-13; Shin et al., 2004, Nat Rev Mol Cell Biol 5:727-738); however, the details of the underlying mechanisms or the specific proteins involved in such regulation remain largely unclear. The significant link between splicing defects and human diseases underscores the paramount importance for understanding the mechanisms of splicing, including the signaling pathways that regulate global splicing as well as splicing of specific subsets of transcripts.
Alternative splicing allows for a single gene to express different isoforms of mRNA, thus playing a major role in contributing to the cellular complexity in higher eukaryotes without the need to expand the genome (Blencowe, 2006, Cell 126:37-47). Global surveying of the human transcriptome estimates that up to 95% of multiexon genes undergo alternative splicing (Pan et al., 2008, Nat Genet 40:1413-1415; Wang et al., 2008, Nature 456:470-476). Importantly, these events are highly regulated by numerous splicing factors in a tissue type-, developmental stage-, and signal-dependent manner. Aberrations in splicing due to mutations in the pre-mRNA are responsible for up to 15% of inherited diseases (Krawczak et al., 1992, Hum Genet 90:41-54). In addition, defects in the splicing machinery itself, due to the loss/gain of function of splicing factors or their relative stoichiometry, are causes of a wide range of human ailments, ranging from cancer to neurodegenerative diseases (Cooper et al., 2009, Cell 136:777-793; Garcia-Blanco et al., 2004, Nat Biotechnol 22:535-546; Licatalosi et al., 2006, Neuron 52:93-101; Venables, 2004, Cancer Res 64:7647-7654). It has been established that splicing is subject to regulation by upstream signaling pathways. However, the details of the underlying mechanisms or the specific proteins involved in such regulation remain largely unclear. The significant link between splicing defects and human diseases underscores the paramount importance of understanding the mechanisms of splicing, including the signaling pathways that regulate general splicing as well as splicing of specific subsets of transcripts.
Small molecules have been essential in uncovering the mechanisms, regulations, and functions of many cellular processes, including DNA replication, transcription, and translation. While several recent reports have described screens for effectors of splicing, only a small number of constitutive or alternative splicing inhibitors have been identified (Kaida et al., 2007, Nat Chem Biol 3:576-583; Kotake et al., 2007, Nat Chem Biol 3:570-575; Levinson et al., 2006, RNA 12:925-930; Muraki et al., 2004, J Biol Chem 279:24246-24254; Pilch et al., 2001, Cancer Res 61:6876-6884; Soret et al., 2005, Proc Natl Acad Sci USA 102:8764-8769; Stoilov et al., 2008, Proc Natl Acad Sci USA 105:11218-11223; Sumanasekera et al., 2008, Biochem Sac Trans 36:483-490).
There is need in the art for a novel means to identify novel modulators of splicing or splicing dependent processes. The present invention fulfills this need.